Antisense Oligodeoxynucleotides
The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds that specifically hybridize to it is generally referred to as “antisense”. The functions of DNA to be interfered with include replication and transcription. The functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of the target gene. In the context of the present invention, “modulation” means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene. In the context of the present invention, inhibition is the preferred form of modulation of gene expression and mRNA is a preferred target.
It is preferred to target specific nucleic acids for antisense. “Targeting” an antisense compound to a particular nucleic acid, in the context of this invention, is a multistep process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. The targeting process also includes determination of a site or sites within this gene for the antisense interaction to occur such that the desired effect, e.g., detection or modulation of expression of the protein, will result. Within the context of the present invention, a preferred intragenic site is the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene. Since, as is known in the art, the translation initiation codon is typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the “AUG codon,” the “start codon” or the “AUG start codon”. A minority of genes have a translation initiation codon having the RNA sequence 5′-GUG, 5′UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, the terms “translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions.
It is also known in the art that a translation termination codon (or “stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively). The terms “start codon region” and “translation initiation codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation initiation codon. Similarly, the terms “stop codon region” and “translation termination codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation termination codon.
The open reading frame (ORF) or “coding region,” which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Other target regions include the 5′ untranslated region (5′UTR), known in the art to refer to the portion of an mRNA in the 5′ direction from the translation initiation codon, and thus including nucleotides between the 5′ cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene, and the 3′ untranslated region (3′UTR), known in the art to refer to the portion of an mRNA in the 3′ direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3′ end of an mRNA or corresponding nucleotides on the gene. The 5′ cap of an mRNA comprises an N7-methylated guanosine residue joined to the 5′-most residue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA is considered to include the 5′ cap structure itself as well as the first 50 nucleotides adjacent to the cap. The 5′ cap region may also be a preferred target region.
Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as “introns,” which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as “exons” and are spliced together to form a continuous mRNA sequence. mRNA splice sites, i.e., intronexon junctions, may also be preferred target regions, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred targets. It has also been found that introns can also be effective, and therefore preferred, target regions for antisense compounds targeted, for example, to DNA or pre-mRNA.
Once one or more target sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.
Antisense compounds are commonly used as research reagents and diagnostics. For example, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes. Antisense compounds are also used, for example, to distinguish between functions of various members of a biological pathway. Antisense modulation has, therefore, been harnessed for research use.
The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. Antisense oligonucleotides have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans.
Antisense oligodeoxynucleotides (ODNs) provide a means to specifically inhibit synthesis of distinct proteins within a cell. For a review, reference is made to Uhlmann, E. and Peyman, A., (1990) Antisense oligonucleotides: a new therapeutic principle, Chem. Rev., 90, 543, incorporated herein in its entirety. Synthetic antisense oligodeoxynucleotides (ODNs) represent a new tool for the discovery of physiological mechanisms in cell cultures, in tissues and in vivo. Ideally, an antisense ODN is targeted in a sequence-specific manner to nucleic acids to offer the exciting possibility of selectively blocking the expression of a particular gene, and then preventing the translation of messenger RNA (mRNA) into protein without changing the expression of other genes (Askari F K, McDonnell W M 1996 Antisense-oligonucleotide therapy. N Engl J Med 334:316-318.). Although the spectrum of actions of antisense ODNs is probably not totally established, hybridization arrest of translation is the most obvious (Helene C, Toulme J J, 1990 Specific regulation of gene expression by antisense, sense and antigene nucleic acids. Biochim Biophys Acta 1049:99-125.). Translational arrest, the antisense ODN interference in protein synthesis, is possible via different pathways that are determined by the target mRNA sequence. One often chosen target is the region upstream and downstream from the initiation codon AUG coding for methionine (Marcus-Sekura C J, 1988, Techniques for using antisense oligodeoxyribonucleotides to study gene expression. Anal Biochem 172:289-295.), but the CAP site and coding region have also been chosen for successful repression of translation. It is assumed that antisense ODNs binding will interfere with the formation of a translation initiation complex and thus block protein synthesis. Another possibility of obstructing gene expression could be by targeting splice junctions and so interfere in RNA maturation from heterogeneous nuclear RNA (hnRNA) to mRNA in the cell nucleus.
RNase H-mediated cleavage of mRNA, facilitated by hybridization to antisense ODN, is widely implicated in antisense ODNs action. RNase H, a ubiquitous enzyme found in both nucleus and cytoplasm, can cleave the target RNA at RNA-DNA oligonucleotide duplexes. RNase H cleavage of mRNA thus reflects an important catalytic activity of the antisense ODNs.
Before an ODN exerts its desired effect on gene expression, it must escape nuclease attack in the extracellular medium, cross the cell membrane, escape the attack of intracellular nucleases, and hybridize with the intended target sequence. Modification of the backbone structure of ODNs renders the ODNs resistant to degradation by nucleases and increases their half-life in biological systems. Phosphorothioate oligodeoxynucleotides are far more popular as inhibitors of specific gene expression, are resistant to degradation by RNases, and are potent at nanomolar concentrations. Cells efficiently take them up, apparently via a saturable and energy-dependent process.
Follicle-Stimulating Hormone
It is known that follicle-stimulating hormone (“FSH”) is required for the maturation of ovarian follicles and testicular spermatogenesis, and that, in adults, circulating FSH regulates gonadal function and steroidogenesis. FSH regulates folliculogenesis in the ovary and spermatogenesis in the testis via specific, high affinity membrane-bound follicle-stimulating hormone receptors (“FSHR”). FSHR number is determined by the steady state equilibrium between receptor synthesis and receptor degradation.
There are numerous chronic diseases that are a function of altered hormonal status, especially the sex hormones. The most dominant of all of the female hormones are the estrogens which control the reproductive system as well as the function of many other cells and tissues, including bones, as well as the cardiovascular and immune systems, angiogenesis, brain and nerves, and lipid metabolism, etc.
The basic factors controlling female ovarian functions are the anterior pituitary gonadotropins: follicle-stimulating hormone (FSH), which directs follicle and ovum development, and luteinizing hormone (LH) that induces estrogen secretion. The hypothalamus controls the pituitary function by means of secreting pulsatile gonadotropin releasing hormone (GnRH) production. There is a strong negative feedback inhibition of hypothalamus/pituitary function that is conducted by estrogen and inhibin (the latter is a glycoprotein that selectively inhibits FSH secretion).
In puberty the hypothalamic GnRH secretion is raised and this induces estrogen production through pituitary LH. Menarche, the first menstrual period, is delayed for about one to one and one half years, and the early menstrual cycles are usually not accompanied by ovulation, which may be delayed for one to one and one half years. During this overall time of 2 to 3 years there is no established feedback mechanism, either positive or negative. As a result hormonal imbalances are induced and a number of physical and psychological disorders manifest themselves.
Premenstrual tension is exhibited by a series of symptoms that occur during the second, luteal phase of the menstrual cycle. Premenstrual tension is induced by a surge of estrogen that arises because the negative feedback inhibition is altered.
Premenstrual syndrome (PMS) or premenstrual tension is a disorder that affects menstruating women one to two weeks before menses begins. The pathophysiologic mechanisms of PMS are weakly understood. One of the causes is a hormonal imbalance, an excessive estrogen level and an inadequate progesterone level. Estrogen levels in the blood and hypothalamus increase at the end of the first part of the menstrual cycle (the follicular phase); the second peak comes a week before a menstrual flow (the luteal phase). It is the second estrogen peak, which coincides with the progesterone peak, that determines the extent of the PMS.
The clinical diagnosis of PMS involves a combination of physical and behavioral symptoms including headache, breast tenderness, swelling of extremities, tension, anxiety and mood swings. It is possible to differentiate women with three premenstrual symptom severity patterns: premenstrual syndrome (PMS) proper, premenstrual magnification (PMM), and low symptom (LS).
Epithelial Ovarian Cancers
Approximately 24,000 new cases of epithelial ovarian cancer (“EOC”) will be diagnosed each year in the United States. Although the absolute number of cases of ovarian cancer pales in comparison to neoplasms of the breast, lung, or colon, ovarian cancer is distinguished by the grim fact that the mortality rate exceeds 70%. Although combination chemotherapy results in disease regression in almost 80% of patients with advanced stage disease, the development of recurrent disease is a common event that ultimately results in death in the majority of cases.
Epithelial ovarian cancer accounts for 80 to 90 percent of ovarian neoplasms. The factors contributing to the initiation, promotion and progression of EOC are incompletely defined. Basic laboratory, animal model and clinical data support the hypothesis that elevated levels of FSH contribute to the pathobiology of this disease. Evidence to support a role for elevated levels of FSH in the promotion and progression of EOC include:                1. The ovarian surface epithelium, which is the origin of epithelial ovarian cancer, expresses the FSH receptor.        2. Proliferation of the ovarian surface epithelial cells can be induced by FSH        3. GnRH agonists inhibit FSH-induced proliferation of ovarian surface epithelial cells        4. Epithelial ovarian cancer primarily occurs in postmenopausal women, in whom serum FSH levels are consistently and chronically elevated        5. Neoplastic epithelial ovarian cells express the FSH receptor        6. Neoplastic epithelial ovarian cells proliferate in response to FSH        7. GnRH agonists inhibit FSH-induce proliferation of neoplastic epithelial ovarian cells        8. The Wx/Wv mouse exhibits spontaneous development of tubular mesothelial adenomas (a precursor lesion of EOC) in response to elevated FSH levels and the development of these precursor lesions can be consistently inhibited by the administration of GnRH agonists        9. In a heterotransplanted model of human ovarian cancer in the nude mouse, tumor growth is significantly greater in surgically castrated animals as compared to age-matched controls.        10. The administration of GnRH agonists to women with advanced stage EOC is occasionally accompanied by a therapeutic response.        
Epithelial ovarian cancer develops from the ovarian surface epithelium (“OSE”), which is a single, focally stratified layer of modified peritoneal cells that is separated from the underlying ovarian stroma by a distinct basement membrane. The ovarian surface epithelial cell layer is contiguous with the mesothelial cell layer of the peritoneal cavity but these cell populations can be differentiated on the basis of biochemical and functional differences. Histochemical studies have revealed the presence of glycogen and mucopolysaccharides within the cells that cover the surface of the ovary. These cells weakly express E-cadherin and CA-125. In addition, cellular membrane receptors are present for estrogen and gonadotropins. Ovarian surface epithelial cells also exhibit 17-beta-hydroxysteroid dehydrogenase activity.
Unlike the peritoneal mesothelium, the epithelial cell layer of the ovary (e.g. the ovarian mesothelium) exhibits a much higher incidence of malignant transformation. Extra-ovarian primary papillary peritoneal carcinoma accounts for only about ten percent of cases presumed to be of ovarian origin based upon intra-operative findings. Although primary ovarian cancer and extra-ovarian papillary peritoneal cancer can be differentiated pathologically, there are few, if any, differences in the epidemiology of these diseases. The proximity of the ovarian mesothelium to the underlying stroma of the ovary provides a unique microenvironment for intercellular interactions. By virtue of this anatomic relationship, the OSE is subject to the effects of steroid hormones and growth factors produced by the ovarian stroma and germ cells of the ovary as well systemic factors, such as elevated levels of FSH.
Epithelial ovarian cancer is generally a disease of postmenopausal women. The incidence of epithelial ovarian cancer ranges from 15.7 per 100,000 women in the 40 to 44 age group to 54 per 100,000 women in the 75 to 79 age group. The mean age at diagnosis is 52 and the disease is uncommon prior to the age of 40. With increasing age, there is a clear increase in the incidence of this disease.
Serum FSH levels are chronically elevated in postmenopausal women, which corresponds to the predominant time of occurrence of EOC. This association has led to the suggestion that elevated gonadotropin levels may play a role in the pathogenesis of this disease. A stimulatory effect of gonadotropins upon the OSE is suggested by the more common occurrence of benign papillary excrescences of the ovarian surface epithelium in postmenopausal as compared to premenopausal women. Additional indirect evidence for a tumorigenic influence of FSH is the observation that patients with gonadal dysgenesis (who have elevated gonadotropin levels) are prone to develop malignant gonadal tumors.
It is well documented that elevated gonadotropin levels are associated with ovarian cancer in most cases, yet serum FSH levels (but not LH) are actually lower in postmenopausal women with ovarian cancer as compared to matched, postmenopausal controls. These data suggest the presence of a tumor-specific inhibitor of FSH release that results in lower serum FSH values in women with ovarian cancer. Inhibin, an ovarian peptide belonging to the TGF beta superfamily, is a potential candidate for this role. Inhibin levels are elevated in some but not all women with advanced stage ovarian cancer. Among epithelial ovarian carcinoma, mucinous cystadenocarcinoma is more likely to be associated with an increased inhibin value than the other histological subtypes.
There is a relationship between serum gonadotropin levels and estrogen replacement therapy (ERT) that merits attention. Women treated with estrogen replacement therapy (ERT) have reduced FSH levels compared to women who do not take ERT. At least one study has suggested that the lifetime risk of EOC is reduced in patients prescribed ERT compared to controls although other studies refute this observation. In one large prospective study of 240,073 peri and postmenopausal women with a seven-year period of follow-up, ever use of estrogen was associated with a rate ratio for fatal ovarian cancer of 1.15 (C10.94-1.42). The risk increased with the duration of estrogen replacement therapy.
Apart from an effect upon serum FSH levels, estrogens also exert a direct effect upon normal and neoplastic OSE cells. Normal OSE cells are estrogen receptor positive. Epithelial ovarian cancers express the estrogen receptor in 70% of cases and the progesterone receptor in 40% of cases. In vitro, 17 beta-estradiol stimulates the proliferation of some but not all estrogen receptor positive cell lines. Estrogens also exert a direct effect upon the ovarian stroma and influence the synthesis of growth factors. Although these observations would suggest that ERT is contraindicated in women with ovarian cancer, there are no published clinical data that suggest that estrogen therapy adversely influences the clinical course of EOC. In fact, ERT may be beneficial by reducing serum gonadotropin levels. It has also been reported that estrogen reduces tumor angiogenesis as well as the invasive potential of neoplastic cells and cell motility in vitro.
Anti-estrogens, such as tamoxifen, inhibit the growth of normal OSE cells as well as the growth in vitro of estrogen receptor positive ovarian cancer cells. However, the antiproliferative effect of tamoxifen is not restricted exclusively to cells that express the estrogen receptor. Neoplastic OSE cells exhibit tamoxifen binding sites that are distinct from the estrogen receptor and occur in greater numbers than those found on normal OSE cells. In clinical studies, disease stabilization or regression has been reported in approximately 17-18% of women with advanced stage disease treated with tamoxifen as salvage therapy.
The FSH receptor is expressed by OSE and EOC. Gonadotropin receptors for both FSH and LH are present on normal and malignant OSE cells. FSH stimulates the proliferation of FSH-receptor positive cells. When FSH binds to its receptor on a normal OSE cell, proliferation is stimulated. FSH also causes an increase in cell proliferation of some, but not all, ovarian carcinoma cell lines. GnRH agonists decrease the proliferation of FSH receptor positive cells in vitro.
Animal models of epithelial ovarian cancer suggest a role for FSH in ovarian tumorigenesis. The hypothesis that elevated gonadotropins play a role in the promotion of EOC is particularly supported by experiments conducted using the Wx/Wv mouse. The B6C3F1 Wx/Wv strain of mice are born with less than 1% of the normal oocyte complement and exhibit rapid oocyte depletion in early life that is accompanied by a concomitant four fold rise in FSH levels. Accompanying this change is the development of complex tubular mesothelial adenomas in 100% of the animals by 4 to 5 months of age. Tubular mesotheliomas are derived from the ovarian surface epithelium and are considered a precursor lesion of EOC.
When Wx/Wv mice are treated with a GnRH agonist, tubular mesotheliomas do not develop. Blaaker et al (1995) compared tumor development was compared in the control group versus a group of Wx/Wv mice treated with a GnRH agonist (Zoladex). All 15 Wx/Wv mice that received sham injections developed bilateral tubular adenomas from the OSE but none of the 11 GnRH-treated animals developed any tumors (Blaaker J et al. Gonadotropin-releasing hormone agonist suppression of ovarian tumorigenesis in mice of the Wx/Wv genotype. Biol Reprod 53:775, 1995).
In a related animal model, mesothelial adenoma formation is also observed when mouse oocytes are destroyed by gamma irradiation early in life. However, mesothelial adenomas do not develop in hypogonadal mice deficient in both GnRH and gonadotropins following gamma irradiation induced oocyte depletion.
Additional data from Peterson et al also indicate that elevated gonadotropin levels appear to play a role in the pathobiology of ovarian cancer. When compared to endocrinologically intact animals, surgically castrated nude mice exhibited significantly greater growth of subcutaneously transplanted human ovarian epithelial carcinoma (BG-1). When surgically castrated nude mice were treated with a long-acting gonadotropin-releasing hormone agonist, the growth of the heterotransplanted tumors was decreased (Peterson C M et al. A long-acting gonadotropin releasing hormone agonist inhibits the growth of a human ovarian epithelial carcinoma (BG-1) heterotransplanted in the nude mouse. Obstet Gynecol 76:264, 1990).
A study by Schiffenbauer et al., further supports of a role for FSH in ovarian tumorigenesis (Schiffernbauer Y S, Abramovitch R, Meir G et al. Loss of ovarian function promotes angiogenesis in human ovarian carcinoma. PNAS 94:13203-8, 1997). Exogenous gonadotropins stimulated the growth of ovarian carcinoma spheroids implanted in nude mice. Tumor growth was accompanied by neovascularization and elevated serum levels of vascular endothelial growth factor (VEGF). The investigators postulated that elevated gonadotropin levels might facilitate the activation of dormant or subclinical disease. If this were the case, a reduction in serum FSH levels following first line chemotherapy when the patient is clinically free of disease would be of potential therapeutic benefit.
Clinical studies of GnRH agonists have demonstrated a therapeutic effect. Several investigators have reported that reduction in FSH and LH by the administration of LHRH agonists can induce a 20-50% partial remission or stable disease in patients who relapse after failing conventional therapies. Prospective, randomized clinical studies of GnRH agonists in combined with chemotherapy have not confirmed the encouraging observations initially reported in small series and anecdotal case reports. However, the trials, which failed to show a therapeutic benefit from the addition of GnRH agonists, are not truly comparable to the earlier reports in that the patients were earlier in the course of their disease when response rates to chemotherapy are much greater. It is conceivable that any beneficial effect of GnRH agonist therapy was masked by the effect of chemotherapy. In the absence of a prospective, single-agent trial, the issue remains unsettled.
FSH enhances the progression of epithelial ovarian cancers, the predominant form of ovarian cancer. Thus, the compositions of the present invention are useful for blocking FSH action on epithelial ovarian cancer cells, which would limit or prevent the progression of the disease. The FSHR antisense oligodeoxynucleotides of the present invention can specifically block FSH action because it has been shown to inhibit synthesis of the FSH receptor. It would have no effect on any cell that does not have FSH receptor. Preferably, the antisense compounds are administered in a sustained release depot formulation as a chemopreventive or as a chemotherapeutic for various cancers, especially ovarian cancers.